Posttranslational acetylation of lysine residues is a highly conserved and important modification enabling the cellular calibration of protein function and/or stability resulting in effects ranging from cytoskeletal reorganization to changes in gene expression. (Weinert, B. T. et al. Sci. Signal. 4, ra48 (2011); Choudhary, C. et al. Science 325, 834-840 (2009); Spange, S., Wagner, T., Heinzel, T. & Kramer, O. H. Int. J. Biochem. Cell Biol. 41, 185-198 (2009); Fass, D. M. et al., “Histone Acetylation and Deacetylation,” in Epigenetic Regulation and Epigenomics, ed. Meyers, R. A. (Wiley-Blackwell, Weinheim, 2012)). Histone deacetylases (HDACs) play a key role in maintaining the balance of acetylation states by catalyzing the removal of acetyl groups from the amino groups of acetylated lysine residues. (Fass, D. M. et al., “Histone Acetylation and Deacetylation,” in Epigenetic Regulation and Epigenomics, ed. Meyers, R. A. (Wiley-Blackwell, Weinheim, 2012)). As a result, these enzymes have become important therapeutic targets for a number of disease states including, but not limited to, cancer and psychiatric illnesses. (Acharya, M. R., Sparreboom, A., Venitz, J. & Figg, W. D. Mol. Pharmacol. 68, 917-932 (2005); Graff, J. & Tsai, L.-H. Annu. Rev. Pharmacol. Toxicol. 53, 311-330 (2013)). As their name implies, HDACs were thought to be primarily responsible for the deacetylation of histones; however, it has become apparent that a large number of non-histone proteins are substrates for these enzymes as well. (Choudhary, C. et al. Science 325, 834-840 (2009); Glozak, M. A., Sengupta, N., Zhang, X. & Seto, E. Gene 363, 15-23 (2005)). The HDAC family is comprised of the NAD(+)-dependent sirtuins (class III) and the metal-dependent HDACs, which can be further divided into three classes (class I: HDACs 1, 2, 3, and 8, class II: HDACs 4, 5, 6, 7, 9, 10, and class IV: HDAC11) based on phylogenetic similarity with class I being localized primarily in the nucleus and classes II and IV shuttling between the nucleus and the cytoplasm. (Gregoretti, I. V., Lee, Y.-M. & Goodson, H. V. J. Mol. Biol. 338, 17-31 (2004); Fass, D. M. et al., “Histone Acetylation and Deacetylation,” in Epigenetic Regulation and Epigenomics, ed. Meyers, R. A. (Wiley-Blackwell, Weinheim, 2012)).
Identification of the endogenous substrates of HDAC enzymes is a fundamental area of HDAC research, and this problem has been particularly acute for the class I enzyme HDAC8. Of all the HDACs, HDAC8 is arguably the best characterized structurally. (Buggy, J. J., Sideris, M. L., Mak, P., Lorimer, D. D., McIntosh, B., Clark, J. M. (2000) Cloning and characterization of a novel human histone deacetylase, HDAC8. Biochem. J. 350, 199-205.; Estiu, G., West, N., Mazitschek, R., Greenberg, E., Bradner, J. E., Wiest, O. (2010) On the inhibition of histone deacetylase 8. Bioorg. Med. Chem. 18, 4103-4110; Tang et al., Bioorganic & Medicinal Chemistry Letters 21 (2011) 2601-2605; Galletti et al., ChemMedChem 2009, 4, 1991-2001; KrennHrubec et al., Bioorganic & Medicinal Chemistry Letters 17 (2007) 2874-2878; Suzuki T., et al., J. Med. Chem. 2012, 55, 9562-9575; Bieliauskas A V et al., Chem Soc Rev. 2008 37(7), 1402-1413). It was the first human class I HDAC structure to be reported, and since then, over 25 additional structures bound to various classes of small molecule ligands and peptides have been disclosed (www.pdb.org). (Wolfson, N. A., Pitcairn, C. A., Fierke, C. A. (2012) HDAC8 substrates: Histones and beyond. Biopolymers 99, 112-126). However, despite this knowledge, few of the enzyme's natural substrates have been identified. (Wolfson et al.) To date, only two cellular substrates of HDAC8 have been identified, namely, the estrogen-related receptor alpha (ERR-α) and the structural maintenance of chromosome 3 (SMC3) protein, of which the latter plays a prominent role in Cornelia de Lange syndrome. (Wilson, B. J., Tremblay, A. M., Deblois, G., Sylvain-Drolet, G., Giguère, V. (2010) An acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha Mol. Endocrinol. 24, 1349-1358; Deardorff, M. A. et al., (2012) HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature 489, 313-317).
It remains unclear which, if any, specific histone residues serve as viable substrates for this isoform. In terms of biological function, HDAC8 has been implicated in various cancers including neuroblastoma, urothelial, and breast cancer as well as in neural crest development. (Oehme, I. et al., (2009) Histone deacetylase 8 in neuroblastoma tumorigenesis. Clin. Cancer Res. 15, 91-99; Niegisch, G. et al., (2013) Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers. Urol. Oncol-Semin. Ori. 31, 1770-1779; Park, S. Y. et al., (2011) Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer. Oncol. Rep. 25, 1677-1681; Haberland, M., Mokalled, M. H., Montgomery, R. L., Olson, E. N. (2009) Epigenetic control of skull morphogenesis by histone deacetylase 8. Genes Dev. 23, 1625-1630). The HDAC8 substrates that mediate these effects are currently unknown.